Multi-purpose blood diluent and lysing agent for differential determination of lymphoid-myeloid population of leukocytes

ABSTRACT

An isotonic multipurpose blood diluent, and a method for use of this diluent with a weak lysing reagent system which is especially suitable for routine enumeration of traditional hemogram values, and also the determination of lymphoid-myeloid populations of leukocytes, particularly in automatic particle counting systems. 
     This blood diluent is capable of affording accurate, reproducible test results. It is an osmotically balanced aqueous solution of preselected pH containing Procaine hydrochloride for maintaining erythrocyte morphology during operation, N-(2-acetamido)iminodiacetic acid (ADA) as a blood cell stabilizing agent, and bacteriostatic agents including sodium 1-hydroxypyridine-2-thione, dimethylolurea and chlorhexidene diacetate which, together with the ADA, allow preferential determination of myeloid-lymphoid leukocytes, and other hematological values. 
     The lysing agent is a mixture of an aqueous solution of at least one quaternary ammonium salt having surface active properties, and an alkali metal cyanide.

BACKGROUND OF THE INVENTION

This invention concerns a blood diluent especially suitable for use inelectronic enumeration and sizing of blood cells, determination ofhemoglobin and their collective indices and platelet parameters in asingle blood cell sample by means of suitable electronicinstrumentation. The diluent comprises a stable water solution ofchemical salts providing an electrolytic solution capable of conductingcurrent to which a blood sample can be added so as to dilute the redblood cells, white blood cells, platelets and other blood components andenable the desired parameters of these blood components to be measured,counted and evaluated.

It is a common medical diagnostic procedure to analyze and test a bloodsample of a patient in order to make certain classic determinations withrespect to the blood sample. This procedure is an important tool for thephysician. Six characteristically important parameters are referred toas red blood cell count (RBC), the hematocrit (HCT), the hemoglobin(HGB), the mean corpuscular volume (MCV), the mean corpuscularhemoglobin (MCH), and the mean corpuscular hemoglobin concentration(MCHC). A seventh important determination is white blood cell count(WBC).

Much effort has been devoted to the development of satisfactorilyautomated leukocyte differential systems. However, a need exists forreagent systems which will be easily adaptable to automatic bloodcounting instruments. In particular, it is desirable to develop reagentsand methods for use with the Coulter Counter® Model S Plus automatedblood counter, manufactured by Coulter Electronics, Inc. of Hialeah,Fla., which will enable the cell volume data accumulated on a CoulterChannelyzer® to discriminate two populations of leukocytes: (1) alymphoid (lymphocytes) population, and (2) a myeloid (neutrophils,monocytes, eosinophils, and basophils) population. Such data are usefulas a screening tool for spotting abnormal leukocyte ratios. Abnormalsituations flagged out by this method give information of diagnosticsignificance, and for further study.

Separation of normal human leukocytes by volume distribution was firstdocumented by Gauthier and colleagues, (Gauthier, J., Harel, P.,Belanger, C. and Fraysse, J., Can. Med. Assoc. J. 97, 793, (1967) andVan Dilla and colleagues, (Van Dilla, M. A., Fulwyler, M. J. and Boone,I. U., Proc. Soc. Exp. Biol. Med. 125, 367, in 1967 as a possibleclinical diagnostic method utilizing the principle of counting andsizing developed by Wallace H. Coulter and employed in Coulter Counter®instruments. These methods were based on the fundamental property of allliving cells to regulate their cell volume by genetic code information.Each type of cell in the circulating blood has its own characteristicvolume ranging from as small as 3 cubic microns for platelets to 450cubic microns for polymorphonuclear cells. Advanced Coulter Counter®instruments have been designed to make use of this volume differentialfor the purposes of counting and determining the size distribution ofplatelets and erythrocytes to detect and monitor pathological states.

Electrical sizing of particles in suspension by a Coulter Counter® typeinstrument has been previously described and documented by many clinicalhematology investigators. It is well known that the form and size of theelectrical pulse generated by a particle passing through a definedelectrical field is influenced by several factors, including size, shapeand conductance of the particles being counted. In blood cellpreparations diluted in an isotonic salt solution, conductivity of thecell membrane is far lower than conductivity of the diluent, andtherefore, blood cells may be considered to be electricallynon-conducting for practical considerations.

Erythrocytes and the lymphoid leukocytes unfortunately overlapconsiderably in cell size, and it is not possible to count one in thepresence of the other by size discrimination alone. Traditional practiceinvolves the use of a strong lytic-surfactant reagent that stromatolyzesthe erythrocytes, reducing them to very small particles or causingmembrane solubilization, and strips the cytoplasm from both the lymphoidand the myeloid leukocytes, leaving only the lyse-resistant nuclei to becounted. Since original cell volume is drastically affected and reducedto a minimum, only a single population is visible by size distributionanalysis. Experimental results indicate that shape changes in leukocytesare not as pronounced as shape changes in erythrocytes; action of alytic agent reduces the leukocyte deformability even further.

The Coulter Counter® Model S Plus automated blood cell counter isdesigned to dilute a sample of whole blood in an isotonic diluent, add alysing agent, and then begin counting after 7.5 seconds. Data arecollected for 4 seconds for erythrocytes and leukocytes and up to 20seconds for platelets. Thus, a diluent-lysing system must provideerythrocyte lysing kinetics sufficiently rapid to effect completestromatization during the lysing period, but not completely strip theleukocytes during this time. In addition, changes in leukocyte volumemust be minimal during the data collection step, and ideally should bestable for several minutes. The reagent system must also preserve theintegrity of the erythrocyte and platelet number and size distribution,and hemoglobin absorbance curve and the total leukocyte count. Fingerstick bloods must be stable when pre-diluted in the isotonic diluent forat least two hours.

To achieve an analysis of the relative populations of lymphoid andmyeloid cells in the blood, the leukocyte volume histogram must showcleanly separated lymphoid and myeloid peaks, with little erythrocytedebris, allowing valleys very close to the baseline. Integration of eachpeak will give the relative populations of the lymphoid and myeloidcells. The lymphoid peak has been demonstrated to contain lymphocytesand small atypical lymphocytes, while the myeloid peak containspolymorphonuclear cells, bands, monocytes, eosinophils, basophils andlarge atypical lymphocytes.

In U.S. Pat. No. 3,874,852 (1975) to Coulter Diagnostics, Inc., aformula is included for a composition containing quaternary ammoniumsalt detergent and cyanide to be employed as a lysing andchromagen-forming reagent for obtaining a single volume leukocyte countand hemoglobin determination in the Coulter Counter® Model S. Furtherinvestigation was required to use quaternary ammonium salts as lysingagents for obtaining the two-population leukocyte count.

In copending patent application Ser. No. 096,697 filed Nov. 23, 1979,now U.S. Pat. No. 4,286,963 to Coulter Electronics, Inc. a lytic diluentfor the rapid lysing of red blood cells in whole blood for making adifferential determination of lymphoid/myeloid populations ofleukocytes, and also measuring hemoglobin by chromagen formation,contains a mixture of an aqueous saline solution of at least onequaternary ammonium salt having surface acting properties, and certainadditives such as 2-phenoxyethanol.

It is known that two volume distribution analysis is difficult becausewith many diluents, the two populations rapidly move into one. There isnot enough time within which to make the computations for analysis.

SUMMARY OF THE INVENTION

The present invention relates to an isotonic multipurpose blood diluent,and a method for use of this diluent with a lysing reagent system toallow routine enumeration of traditional hemogram values, and also thedisplay and calculation of the lymphoid/myeloid histogram of theleukocytes and their relative concentrations, particularly in automaticcounting systems such as the automated Coulter Counter® equipment withunmodified programming and an external or internal leukocyteChannelyzer® instrument capability.

This multipurpose isotonic blood diluent comprises a cell stabilizingmixture of organic buffers, anesthetics, and germicides in anosmotically balanced and substantially neutral solution, and serves toslow the kinetics of cytoplasmic stripping from the leukocytes, whilestabilizing the traditional hemogram parameters. More specifically, theisotonic diluent is an aqueous solution of:

1. N-(2-acetamido)iminodiacetic acid (ADA),

2. Procaine hydrochloride,

3. Chlorhexidene diacetate,

4. Dimethylolurea,

5. Sodium 1-hydroxypyridine-2-thione,

6. Sodium sulfate and sodium chloride

the diluent being adjusted to a pH 7.0±0.1 with sodium hydroxide orhydrochloric acid solution, as necessary, and to an osmolality of 320±5milliosmoles per kilogram with sodium chloride.

The lysing reagent is a mixture of an aqueous solution of at least onequaternary ammonium salt having surface active properties, and an alkalimetal cyanide. Presentation of data may be accomplished using standardCoulter Counter® equipment in conjunction with a Channelyzer® and an X-Yplotter. Ancillary calculating and data handling devices are desirablefor complete automation, but are not essential to performance of themeasurements.

DETAILED DESCRIPTION OF THE INVENTION Description of the PreferredEmbodiments

The preferred formulation of the isotonic diluent is:

    ______________________________________                                                              Effective                                               Ingredient            Concentration Range                                     ______________________________________                                        Procaine hydrochloride                                                                              0.11 g/L                                                N-(2-acetamido)iminodiacetic acid (ADA)                                                             1.40 g/L                                                Chlorhexidene diacetate                                                                             0.02 g/L                                                Dimethylolurea        1.00 g/L                                                Sodium 1-hydroxypyridine-2-thione                                                                   0.50 g/L                                                Sodium hydroxide      0.50 g/L                                                Sodium sulfate, anhydrous                                                                           9.72 g/L                                                Sodium chloride       4.50 g/L                                                Water                 Sufficient for 1 liter                                  ______________________________________                                    

This diluent is adjusted to pH 7.0±0.1 with sodium hydroxide orhydrochloric acid solution as necessary. The osmolality is adjusted to320±5 milliosmoles per kilogram with sodium chloride.

The chemical and biological action of Procaine hydrochloride, sodiumsulfate and sodium chloride is disclosed in U.S. patent application Ser.No. 936,570 filed Aug. 22, 1978 now U.S. Pat. No. 4,213,876. It is wellknown that blood cells contain ATPase enzymes that transport alkalimetal cations associated with small anions (e.g. chloride) into and outof the cell by an energy-requiring mechanism in order to maintainosmotic balance, tergidity of the cell and proper membrane potentials.Sodium ions associated with the much larger sulfate anion are apparentlynot transported as readily due to greater differences in charge densitybetween sulfate and chloride anions and the potential change in membranecharge when ions are transported into or out of the cell. Procainehydrochloride, as one of a class of anesthetics, has been reported tohave a stabilizing influence on the erythrocyte membrane, but the modeof action is uncertain. Seeman (P. Seeman, Biochemical Pharmacology 15,1753, 1966) suggests that the hydrophobic portion of the Procainemolecule dissolves in the membrane, leaving the polar (charged) and nearthe outer surface, and causes expansion of the membrane as shown byelectron microscopy. It is highly probable that this membrane expansionis due at least in part to changes in the membrane potential induced bythe anesthetic and reinforced by salts of low ionic mobility. Balance ofinternal osmotic pressure within the cell is maintained by the inclusionof approximately 30 milliosmoles of sodium chloride. Sodium sulfate isalso well known for its ability to solubilize abnormal plasma globulinsand to reduce or eliminate turbidity in the hemoglobin solution due toelevated leukocyte counts.

The N-(2-acetamido)iminodiacetic acid (ADA) has previously beendescribed as a ligand for metal cations (U.S. Pat. No. 4,116,635,Jaeger) and as an organic buffer. Its use in the present invention ispredicated on its ability to assist weak quaternary ammonium salt lysingagents in reducing lysed erythrocyte debris to particle sizeselectrically smaller than 45 cubic microns, thus preventing erythrocyteinterference with the enumeration and distribution of leukocytes intotwo distinct populations (lymphoid and myeloid). In addition, ADA hasbeen found to help stabilize the size distribution, cellular shape, andmost importantly the high degree of cellular dispersion of erythrocytesand platelets to an extent not previously observed with other compounds.Although the mechanism of action of ADA has not been rigorouslyinvestigated, it is known to be a moderately strong ligand for group IIalkali metals and group IIB transition metals, as well as a reasonablyefficient buffer. Resembling an amino acid to a strong degree, ADAapparently is attracted to and interacts with the cell membraneproteins. It is then coordinated with metal cations giving the outermembrane surface a more positive charge "appearance" to the surroundingsolution, thus encouraging solvation and anion attraction around theouter membrane. This coating of solvent molecules and anions effectivelyprevents approach and agglutination interaction with other cells insuspension. The buffering action of the ADA prevents changes in this"local" environment, precluding desolvation and loss of stability.

This mechanism, coupled with the action of Procaine and sodium sulfate,assists in the stabilization of the cellular components of the bloodsample, rendering them essentially unchanged from their original statein the blood specimen in terms of number, size distribution and shape.This factor is of great diagnostic importance in the interpretation ofthe traditional hemogram parameters.

Chlorhexidene diacetate, though usually used as a virocide andgermicide, is only marginally effective by itself at the concentrationused, but significantly aids ADA in reducing the background debris thatinterferes with leukocyte size distribution enumeration. Chlorhexidenealso bears some resemblance to the amines arginine and guanidine andmight be expected to interact with membrane proteins by coordination andhydrogen bonding. As such, it apparently aids the lysing agents tostromatolyze completely the erythrocyte membranes.

Dimethylolurea, a condensate of formaldehyde and urea, is believed toreact very slowly in solution at neutral pH with the leukocyte membranesto afford a measure of stability upon standing that is not evident uponremoval of this compound. It is known as a bacteriostatic antisepticcompound, preventing growth of microorganisms at moderateconcentrations. Other compounds which were joined to stabilizeleukocytes are hexamethylenetetramine, and N-hydroxymethylacetamide.

Sodium 1-hydroxypyridine-2-thione (U.S. Pat. No. 2,745,826, OlinMatheson 1956) has been used as fungicide and bacteriocide in dandruffshampoos (U.S. Pat. Nos. 3,236,733 and 3,281,366, Procter and Gamble1966) and is used here as a bacteriocide and fungicide that exhibits noapparent deleterious effect on the shape, size distribution or number ofthe cellular components in whole human blood.

The use of both dimethylolurea and sodium 1-hydroxypyridine-2-thionegives a very bacteriocidal product which is superior to that obtainablewith either substance used alone. This combination gives a stablehemogram for both normal and abnormal samples while simultaneouslyallowing volume distribution information about white cells to becollected by an automated instrumentation system.

It is a primary aim of this diluent system to suitably stabilize cellsize, shape and integrity of all blood cellular components to an extentnot previously achieved in order to promote diagnostic accuracy of bloodhemograms derived from automated volume distribution analysis andenumeration. The present invention allows the extension and improvementof prior arts by rendering the blood stream conditions of cellsrelatively unchanged when diluted and prepared for automated blood cellcounting and sizing. When used in conjunction with an appropriate lysingagent, prior art may be extended to provide volume distributioninformation about leukocytes.

The described diluent will reproduce accurate hemograms with anysemi-automated or automated Coulter® blood cell counter, but willproduce two volume leukocyte histograms only when used in conjunctionwith the lysing reagent of this invention.

The lysing agent is an aqueous solution of at least one quaternaryammonium salt having surface active properties, and an alkali metalcyanide, as described generally in U.S. Pat. No. 3,874,852 Coulter 1975.However, the effective range and concentration of ingredients statedherein must be followed in order to obtain satisfactory results by themethod of this invention.

In the preferred compositions, the long chain alkyl in the followingformula has 12-16 carbon atoms, the short chains are trimethyl, and X⁻is chloride or bromide.

The quaternary ammonium salt detergent has the formula: ##STR1## whereR₁ is a long chain alkyl radical having 10 to 18 carbon atoms, R₂, R₃and R₄ are short chain alkyl radicals having 1 to 6 carbon atoms, and X⁻is a salt forming radical such as Cl, Br, I, PO₄ and CH₃ SO₄.

A preferred formulation for the lysing agent is:

    ______________________________________                                                             Effective                                                Ingredient           Concentration Range                                      ______________________________________                                        Dodecyltrimethylammonium chloride                                                                  60 g/L 40-70 g/L                                         50% solution                                                                  Tetradecyltrimethylammonium bromide                                                                6 g/L 4-7 g/L                                            Mytab®                                                                    Potassium cyanide    300 mg/L 250-500 mg/L                                    Water                sufficient for 1 Liter.                                  ______________________________________                                    

The above lysing agent is very much weaker and slower in reaction ratethan the currently used Lyse S II®. Alternate quaternary ammonium saltsthat also are effective include Cetrimide® (hexadecyltrimethylammoniumbromide) and Bretol® (cetyldimethylethylammonium bromide), both aloneand in combination with dodecyltrimethylammonium chloride. The diluentsystem is apparently able to stabilize the leukocyte membranesufficiently to slow the kinetics of the lysing agent reaction to apoint where it is possible to distinguish the smaller volume of lymphoidcells from the larger volume of the myeloid series (neutrophils,monocytes, eosinophils and basophils). Repeated measurement of leukocytesize distribution have shown the histograms to be stable for up to 30seconds before degeneration. Experimental results indicate that lymphoidcells are reduced essentially to their minimum cellular volume withinthe 7.5 second lysing time. The myeloid cells appear two to three timestheir terminal volume for up to 30 seconds after addition of the lysingagent after which the volume is slowly reduced until the myeloidfraction merges with the lymphoid fraction.

Using an erythrocyte calibrated Coulter® Model S Plus and a calibratedCoulter® Model C-1000 Channelyzer® the volumes of the lymphoid andmyeloid peaks after treatment with the reagent system were found to bein the vicinity of 85 cubic microns and 260 cubic microns, respectively,with narrow distribution widths. Leukocytes obtained from fresh wholeblood by simple sedimentation on a Ficoll-Paque® gradient were found toexhibit volumes of approximately 260 cubic microns for the lymphoid peakand 500 cubic microns for the myeloid peak with much wider distributionwidths. A sample of fresh blood separated by centrifugation on aFicoll-Paque® gradient using a standard technique produced mononuclearcells (lymphocytes and monocytes) separated from granulocytes(neutrophils, eosinophils and basophils). The lymphocytes were found tobe coincident with the lymphoid peak at 260 cubic microns while themonocytes were found to be centered around 550 cubic microns. Thegranulocyte fraction formed a rather broad peak centered around 500cubic microns. The separation of the two populations was much moredistinct with the chemically lysed blood from the Coulter Counter® ModelS Plus experiments than with the viable leukocytes obtained from theFicoll-Paque® method. It is, therefore, apparent that the lysing agentdamages the leukocytes substantially, since their volumes are reducedtwo-to-three-fold in comparison to leukocytes obtained from theFicoll-Paque® separation of the same blood sample.

In the present invention incorporation of dodecyltrimethylammoniumchloride has been found to reduce preferentially the volume of thelymphoid cells (i.e. a three-fold reduction in volume from 260 cubicmicrons to 85 cubic microns), while influencing the volume of themyeloid cells to a lesser extent (i.e. a two-fold reduction in volumefrom 500 cubic microns to 260 cubic microns. Dodecyltrimethylammoniumchloride is actually a poor lysing agent itself. It appears to moderatethe strong lytic effects of Mytab®, slowing the lysing kinetics to adegree that allows measurement of the different cell nuclei volumes. Theincorporation of dodecyltrimethylammonium bromide also gives a muchcleaner, faster lysis of the erythrocytes and faster conversion of thehemoglobin with lower concentrations of Mytab®, thus giving betterstability to the leukocyte populations.

Preliminary data on more than one hundred normal and abnormal freshblood specimens obtained from a blood donor center and local hospitalsindicate a high degree of correlation between leukocyte population datafrom the Coulter Counter® Model S Plus-Channelyzer® PG,14 system andmanual 100 cell differentials. Although slight variation in myeloid andlymphoid fractions were observed, no drastic departures from correlationwere noted.

Using the combination of the diluent and lysing reagent of theinvention, spectrophotometric scans (wave length vs. absorbance) of thelysed blood from the leukocyte bath of the Coulter Counter® Model S Plusproduced hemoglobin curves essentially identical to those produced byLyse S®II; however, the absorbance at 540 nm and the resultingcalculated hemoglobin values are approximately 0.2 g/l higher than withthe combination of Isoton II® and Lyse S®II. Mean cell volume valueshave also been noted to be 1.0 to 1.5 cubic microns larger in the newtwo reagent system. All other parameters are virtually identical tothose produced with the combination of Isoton II® and Lyse S®II.

We claim:
 1. A multipurpose isotonic diluent comprising a mixture of anaqueous solution of:1. Procaine hydrochloride, 2.N-(2-acetamido)iminodiacetic acid (ADA),
 3. Chlorhexidene diacetate, 4.Dimethylolurea,
 5. Sodium sulfate and sodium chloride, said diluentbeing adjustedto a pH of 7.0±0.1 with sodium hydroxide or hydrochloricacid solution, as necessary, and to an osmolality of 320±5 milliosmolesper kilogram with sodium chloride.
 2. The isotonic diluent of claim 1including sodium 1-hydroxypyridine-2-thione as an antibacterial agent.3. The isotonic diluent of claim 1 wherein the concentration of saidProcaine hydrochloride is 75 to 250 mg/l.
 4. The isotonic diluent ofclaim 1 wherein the concentration of said N-(2-acetamido)iminodiaceticacid is 1.2 to 2.0 g/l.
 5. The isotonic diluent of claim 1 wherein theconcentration of said chlorhexidene diacetate is 0.01 to 0.03 g/l. 6.The isotonic diluent of claim 1 wherein the concentration of saiddimethylolurea is 0.50 to 1.0 g/l.
 7. A method which comprises the stepsof:I. treating a blood sample with an isotonic diluent comprising anaqueous solution of:
 1. Procaine hydrochloride,2.N-(2-acetamido)iminodiacetic acid (ADA),
 3. Chlorhexidene diacetate, 4.Dimethylolurea,
 5. Sodium sulfate and sodium chloride, said diluentbeing adjustedto a pH 7.0±0.1 with sodium hydroxide or hydrochloric acidsolution, as necessary, and to an osmolality of 320±5 milliosmoles perkilogram with sodium chloride; and then II. lysing with a reagentcomprising a mixture of an aqueous solution of at least one quaternaryammonium salt detergent and an alkali metal cyanide, said quaternaryammonium salt and said alkali metal cyanide being present in aconcentration range which is effective to give a differentialdetermination of lymphoid and myeloid populations of leukocytes followedby determination of hemogram values, particularly in automatic particlecounting systems.
 8. The method of claim 7 wherein sodium1-hydroxypyridine-2-thione is present as additional reagent in Step I.9. The method of claim 7 or 8 wherein said lysing agent in step IIcomprises one or more quaternary ammonium salt detergents selected froma group of compounds having the formula: ##STR2## where R₁ is a longchain alkyl radical having 10 to 18 carbon atoms, and R₂, R₃ and R₄ areshort chain alkyl radicals having 1 to 6 carbon atoms, and X⁻ is a saltforming radical, selected form the group consisting of Cl, Br, I, PO₄,HSO₄ and CH₃ SO₄, and an alkali metal cyanide.
 10. The method of claim 7or 8 wherein said quaternary ammonium salt detergent ishexadecyltrimethylammonium chloride.
 11. The method of claim 7 or 8wherein said quaternary ammonium salt detergent ishexadecyltrimethylammonium bromide.
 12. The method of claim 7 or 8wherein said quaternary ammonium salt detergent isdodecyltrimethylammonium chloride.
 13. The method of claim 7 or 8wherein said quaternary ammonium salt detergent istetradecyltrimethylammonium bromide.
 14. The method of claim 7 or 8wherein said quaternary salt detergent is cetyldimethylethylammoniumbromide.
 15. The method of claim 7 or 8 wherein said quaternary saltdetergent is a mixture of dodecyltrimethylammonium chloride andtetradecyltrimethylammonium bromide or hexadecyltrimethylammoniumbromide.
 16. The method of claim 7 or 8 wherein said quaternary ammoniumsalt detergent is a mixture of 40 to 70 g/l of dodecyltrimethylammoniumchloride (50% solution) and 4 to 7 g/l of tetradecyltrimethylammoniumbromide, and said alkali metal cyanide is 250-500 mg/l of potassiumcyanide.
 17. In a method of determining leukocytes and hemoglobin inblood wherein a lysing agent containing an aqueous solution of aquaternary ammonium salt having surface active properties, and an alkalimetal cyanide, are used to stromatolyze erythrocytes and platelet cellsand to convert hemoglobin to a chromagen, the improvement wherein saidlysing agent is a mixture of 40 to 70 g/l of dodecyltrimethylammoniumchloride (50% solution) and 4 to 7 g/l of tetradecyltrimethylammoniumbromide or hexadecyltrimethylammonium bromide, and said alkali metalcyanide is 250 to 500 mg/l of potassium cyanide.